At least seven sarcomeric myosin heavy chain (MHC) genes are expressed in mammalian skeletal muscles and these are slow type I (cardiac), embryonic (Emb), neonatal (Neo), fast-type IIa, IIx, IIb, and the extraocular (Eo). With the exception of type I MHC gene, skeletal MHC genes are clustered within -400 kilobases on chromosome 10 in the rat (17 in human) in the order Emb, IIa, IIx, IIb, Neo, and Eo (see Fig. 2). This MHC gene organization has been conserved through millions of years of evolution. We propose that the clustered organization of skeletal fast MHC genes is essential for their coordinated regulation in limb muscles. Thus, the main goal of this proposal is to establish whether the fast MHC genes are regulated as a transcriptional unit in a coordinated-fashion involving tandem gene-cross-talk (as illustrated in Fig 1), or whether they are regulated independently. To attain this goal, we will examine skeletal MHC regulation in two types of MHC transition: slow to fast MHC in inactive slow muscle (phase I), and fast to slow in overloaded fast muscles (phase II). We will test the working hypothesis that the cooperative regulation of skeletal MHC genes involves one of three mechanistic schemes: 1) a common set of transcription factors/regulatory elements that up regulates one gene while simultaneously down regulating the other (e.g., type I to IIx);2) alternatively, a common milieu of transcription factors that are temporally controlled by the stimuli thereby affecting the target genes in an antithetical fashion (I to IIx;IIx equilibrium IIb);and 3) a unique cooperative process involving tandemly aligned genes in which the downstream (3') gene regulates the upstream one via an intergenic promoter that transcribes antisense RNA that interferes with posttranscriptional events of the 5'aligned gene (type IIa equilibrium IIx;see figure 1). For each experimental paradigm we will: a) develop a working model on the mode of MHC gene regulation in the transforming muscle using analyses of primary transcripts, antisense RNAs, and mature mRNAs of all the MHC isoforms in the target muscle;b) characterize the transcriptional regulation of the target genes, via both in silico and in vivo analyses of the IIa, IIx, and IIb promoters, including the IIa/IIx and IIx/IIb intergenic regions and c) identify transcription factors involved in this coordinated regulation and test their involvement in regulation by overexpressing them in the context of MHC gene promoter transfections. Collectively, these aims explore a new area of analyses taking advantage of new technologies and tools that were not available until recently. The results will give insight on regulatory mechanisms and cooperative regulation of the MHC gene locus.